Cleavable reagents for specific delivery to disease sites

ABSTRACT

A therapeutic reagent to control one or more reactions of the immune system in a host, or to deliver anti-tumour agents, or disease treatment agents to the host. The therapeutic reagent comprises a regulatory moiety and a carrier protein that inactivates or substantially reduces the activity of the regulatory moiety. Also, the therapeutic reagent includes a cleavage site at which cleavage of the therapeutic reagent can occur to free the regulatory moiety from the carrier protein so that the therapeutic reagent can act at a diseased site in the host.

FIELD OF THE INVENTION

This invention relates to therapeutic reagents that can be manipulatedto have a reduced effect or activity when circulating in the body of ahost, but which are designed to be released in an active form atspecific sites and times when needed. In particular, the inventionrelates to regulators of immune functions such as anti-complementreagents, which may be used to regulate the negative roles of immunemolecules or cells in the host. Further, the invention relates to theuse of said therapeutic reagents, pharmaceutical compositions includingsame and methods of medical treatment.

BACKGROUND OF THE INVENTION

The complement (C) system is an important component of the immune systemof hosts, including humans and animals. C is known to consist of anumber of proteins that act in a proteolytic cascade to target antigensor cells. During this sequence, one protein activated through bindingantigen cleaves the next reacting protein to generate a new activatedproteolytic enzyme, which cleaves and thereby activates the next proteinin the sequence. Proteolytic fragments and protein complexes generatedduring activation have phlogistic activity, and cause inflammatorytissue changes such as increased vascular permeability and attraction ofpolymorphonuclear leukocytes.

Examples of such proteins include C5a, C3a and MAC (cytolyticmacromolecular membrane attack complex). Targeting of a cell by Cresults in cell damage or death directly through formation of MAC, orindirectly through initiation of inflammation due to production of theinflammatory mediators (C5a, C3a and MAC) and phagocytosis of C targetedcells.

To protect themselves from this potentially harmful defence mechanism,cells express, on their surface, complement regulatory proteins (CRegs)which rapidly and efficiently inactivate ‘accidental’ foci of Cactivation (Morgan, B & Harris C., Complement Regulatory Proteins(1999)).

CRegs function either by inactivating enzymes, such as the C3 and C5convertases, which are formed during C activation and which areresponsible for cleavage of C3 and C5, or by interfering with MACformation. In humans, the C regulators membrane cofactor protein(MCP;CD46), decay accelerating factor (DAF;CD55) and complement receptor1 (CR1 ;CD35) inhibit C by accelerating the decay of or (with factor I)irreversibly inactivating the C3 and C5 convertase enzymes. A fourthregulator, CD59, inhibits MAC formation by binding C8 and/or C9, andinhibiting C9 polymerisation during MAC formation.

In normal circumstances, these control mechanisms are sufficient toprotect cells from damage by homologous C. However, evidence of Cactivation is abundant in diverse inflammatory diseases includingrheumatoid arthritis (RA), systemic lupus erythematosus (SLE),glomerulonephritis, adult respiratory distress syndrome (ARDS),ischemia-reperfusion injury, demyelination, myaesthenia gravis, Arthusreaction, rejection in transplantion, lupus nephritis and multiplesclerosis. For example, in RA, soluble products of C activation areabundant in the synovial fluid of affected joints. Complement depositsare evident in synovial tissue, together with leukocytes (neutrophilsand T cells) attracted to the site by a gradient of C5a and otherchemo-attractants.

Whilst C itself is not always the primary cause of disease, it acts tosustain the pro-inflammatory cycle, can exacerbate the disease andperpetuate and extend tissue damage due to non-targeted activity.

DESCRIPTION OF THE PRIOR ART

Research has been carried out to find therapeutic reagents capable ofinhibiting the C cascade and preventing the formation ofpro-inflammatory mediators. Some groups have concentrated onhigh-throughput screening to identify small chemical compounds thatmight inhibit C. Others have exploited the naturally occurringcell-associated CRegs by generation of soluble, recombinant forms ofthese molecules that inhibit C through their native activities. Yetothers have developed antibodies that block activation of specificcomponents in the C system.

Anti-C reagents are known, most of which inhibit production of C5a andMAC, the key active by-products of C activation. The best-describedexamples are, first, a scFv that binds C5 and prevents its enzymaticcleavage, and second, a soluble, recombinant form of CR1 (SCR1) thatinhibits the amplification enzymes in the activation pathways of C. Bothof these reagents have been used in acute conditions, such as adultrespiratory distress syndrome (ARDS), or ischaemia-reperfusion injury,which occurs in many clinical contexts, including myocardial infarctionfollowing cardiopulmonary bypass.

Known reagents, with the exception of sCR1, have low molecular weights,for example, from 12 kDa for CD59 to 40-50 kDa for DAF, MCP and Crry,therefore, these reagents are rapidly cleared from the body via thekidneys.

In addition to development of sCR1 for anti-C therapy, solublerecombinant forms of other human and rodent C regulators have beengenerated and tested in C-mediated inflammatory conditions, such as theArthus reaction and rejection in xenotransplantation. In mostcombinations, human C regulators also inhibit rodent C and rodent Cregulators control human C. For example, it has been shown that humanand rodent DAFs are not species-specific in their complement inhibitingactivities (Harris et al Immunology 100 462-470 (2000)). However,administration of a foreign C regulator results in a prompt immuneresponse in the recipient, limiting its function to just a few days.This has restricted the ability to test human C regulators, such assCR1, in chronic disease models in rodents.

Various modified forms of the C regulators have been produced andtested. Attempts have been made to combine two regulatory activitiesinto one reagent, but these attempts have resulted in linear, inflexiblemolecules where the CRegs are fused end-to-end, making them unsuitablefor targeted action (Higgins et al in J Immunol 158 2872 (1997)). Achimeric molecule, in which mouse Crry has been fused to mouse IgG1domains has been produced (Quigg et al J Immunol 4553 (1998)). This hasbeen used in the therapy of murine glomerulonephritis.

Known reagents, including sCR1, have short half-lives in vivo (minutesto hours), requiring frequent systemic administration and limiting theirroles to the therapy of acute situations. They are not suitable forlong-term use in the treatment of chronic disease.

In the aforementioned paper by Harris et al (2000), human DAF-Ig hasbeen produced, in which DAF is tethered to the Fe fragment of animmunoglobulin molecule. Tests in vitro and in vivo have demonstratedthat Ig fusion proteins, such as DAF-Igs, have anti-C activity.Subsequent in vivo tests have demonstrated the ability of fusionproteins to remain in the circulation and to inhibit plasma C activityover longer periods of time.

A confounding problem with current CReg-based anti-C reagents is that Cactivity is inhibited systemically. Although this may be of littleconsequence in acute situations, long-term reduction in systemic Cactivity is not desirable. This is because long-term inhibition of C maypredispose individuals to infection, and also severely compromise theC-dependent process of immune complex solubilization and clearance.

We set out to overcome the problems of known reagents, specificallyshort half-life in vivo and systemic inhibition of C, by engineering atherapeutic reagent that is long-lived in the circulation and has littleor no C inhibitory activity while in the circulation, but which can beactivated at sites of inflammation and/or C activation. Such an anti-C“pro-drug” would offer advantages over current reagents, which treatacute situations of C activation, and allow for the first time use of ananti-C therapeutic reagent in the treatment of chronic illnesses inwhich C activation is implicated.

We have taken advantage of our observation that, in some CReg-Ig fusionproteins, the C regulator has much reduced or absent C regulatoryfunction. Release of the CReg from the Ig moiety restores C regulatorycapacity. Inclusion of one or more specific enzyme cleavage sitesbetween the CReg and the hinge region of the Ig moiety can provide atherapeutic reagent that can possess the desirable properties of a longhalf-life in vivo, minimal systemic disturbance and efficient Cregulation at the site of inflammation or disease.

SUMMARY OF THE INVENTION

Accordingly, the present invention seeks to provide therapeutic reagentshaving a long plasma half-life, minimal systemic effects and anefficient function at the site of pathology. Delivery to the appropriatesite may be enhanced by the inclusion of targeting elements. Thesereagents include anti-C agents. These therapeutic agents differ frompreviously-described reagents, in that the intact therapeutic reagent or‘prodrug’ is designed to express little or no systemic activity.Instead, sites are engineered between the active agent, which is aregulatory moiety, and a carrier moiety, such as an Ig, whereby theagent is released in an active form at the site of pathology to mediateits therapeutic effect. For example, inhibition of the C cascade atsites of inflammation can be achieved using a prodrug comprising a CRegattached via a cleavable sequence to a Ig Fc domain. The Ig moiety ischosen both to minimise C regulatory function of the attached CReg inprodrug form and to maximise the half-life of the CReg-Ig prodrug in thecirculation. The therapeutic reagents may therefore be viewed asprodrugs when circulating in the body and active drugs following releaseof the CReg or other active agent at the target site.

In addition, the therapeutic reagent preferably further comprises aspecific targeting sequence that can enhance delivery to the site ofdisease.

According to the present invention there is provided a therapeuticreagent to control one or more reactions of the immune system in a host,said therapeutic reagent comprising:

-   -   i. at least one regulatory moiety that is an immunoregulatory        protein (IRP) or a functional fragment thereof;    -   ii. a carrier protein which renders said IRP inactive or        substantially inactive; and    -   iii. positioned therebetween at least one cleavage site;        -   whereby        -   when the regulatory moiety is at, or adjacent, target organ            or tissue in the host, said cleavage site is cleaved,            freeing the regulatory moiety from the carrier protein and            restoring its regulatory activity.

According to a yet further aspect of the invention there is provided atherapeutic reagent that is inactive systemically comprising:

-   -   i. at least one regulatory moiety that has a therapeutic        activity;    -   ii. a carrier protein which renders said regulatory moiety        inactive or substantially inactive; and    -   iii. positioned therebetween at least one cleavage site        characterised in that said cleavage site is a substrate for a        matrix metalloproteinase (MMP) or an aggrecanase; whereby        -   at sites where MMP's or aggrecanase are active in the host            said cleavage site is cleaved and said regulatory moiety is            freed from the carrier protein and so able to perform its            therapeutic function.

According to a yet further aspect of the invention there is provided atherapeutic reagent to control one or more reactions of the immunesystem in a host, said therapeutic reagent comprising:

-   -   i. at least one regulatory moiety that is an immunoregulatory        protein (IRP) or a functional fragment thereof:    -   ii. a carrier protein which renders said IRP inactive or        substantially inactive; and    -   iii. positioned therebetween at least one cleavage site        characterised in that said cleavage site comprises a substrate        for at least one enzyme of the Complement system; whereby        when said therapeutic reagent is at or adjacent a site in the        host where Complement is active said cleavage site is cleaved so        freeing the immunoregulatory moiety from the carrier protein and        enabling it to perform its immunoregulatory activity.

The term “regulatory moiety” is used to mean the part of the therapeuticreagent that acts or causes an effect in the host.

Preferably, the cleavage site is between the regulatory moiety and thecarrier protein. More preferably, the cleavage site is positionedbetween the regulatory moiety/CReg and a hinge region of the carrierprotein/Ig.

It is preferred that the cleavage site comprises an extrinsic moiety atwhich the therapeutic reagent can be cleaved under cleavage conditions.More preferably, it comprises an extrinsic amino acid or proteinsequence, which is inserted between the regulatory moiety and thecarrier protein. By extrinsic, is meant that the amino acid or proteinsequence is not naturally a component of the regulatory moiety or thecarrier protein and must therefore be inserted in the therapeuticreagent together with the regulatory moiety and carrier protein.Ideally, the cleavage site is susceptible to cleavage by enzymes of thematrix mettaloproteinase (MMP) and aggrecanase families. Secondarily, itis susceptible to enzymes of the complement system. It therefore followsthat a therapeutic reagent employing these latter cleavage sites willbecome active at locations where complement is active because complementenzymes will cleave the cleavage site and so release the regulatorymoiety which can then function in a regulatory manner to achieve itstherapeutic effect.

Nevertheless, the cleavage site may comprise intrinsic amino acids orproteins that are already present, for example as part of the carrierprotein or regulatory moiety. Again, the amino acids or proteins may beacted on by enzymes at a particular site so that the regulatory moietyand the carrier protein become cleaved, thereby enabling the therapeuticagent to act at a site in the host's body. However, in either case, itmust be ensured that no cleavage site is present in the therapeuticreagent that would result in significant cleavage thereof to release theregulatory moiety in active form other than at or adjacent the targetorgan or tissue; which, in the instance where the cleavage site issusceptible to cleavage by complement enzymes would be at sites wherecomplement was active.

Preferably the regulatory moiety is an immunoregulatory protein, such asa C regulatory protein (CReg). Alternatively, the regulatory moiety maybe a regulatory protein involved in other types of immune response.Ideally, the CReg is an immunoregulatory protein that acts either as adecay accelerating factor or a cofactor for the plasma protease factor 1or to inhibit formation of membrane attack complex. In one embodiment ofthe invention the regulatory moiety may also be a combination of CRegand other regulatory proteins. It is envisaged that the CReg may be anyof those described (Morgan & Harris in Complement Regulatory Proteins(1999)), including those mentioned above, especially DAF, CD59, Crry,active fragments of CR1 and MCP, and may also include active fragmentsof factor H (FH) or other C regulators. Further, the regulatory moietymay comprise more than one active agent, such as more than one CReg.Alternatively, and more preferably, a therapeutic reagent comprising asingle active agent may be co-administered with another therapeuticreagent containing a single, but different, regulatory moiety.

It is preferred that the carrier protein is an immunoglobulin (Ig) Fcfragment. Suitable Igs include IgG1, IgG2, IgG3 or IgG4, with IgG4 beinga preferred Ig and IgG2 especially preferred. Modifications of the Ig tominimise activity of the prodrug-bound CReg, to extend plasma half-lifeor to minimise effector functions of the Fc are included. In oneembodiment of the invention the Fab arms of the Ig may be replaced bytwo CReg moieties. Ideally, the immunoglobulin is human immunoglobulin.

The therapeutic reagent may comprise an immunoglobulin in which one armcomprises a CReg, while the other may comprises a targeting moiety suchas a Fab specific for a certain cell or tissue or an adhesion moleculespecific for a certain cell or tissue. Targeted reagents may alsoinclude a Fab on one arm of the Ig while the other arm comprises adifferent regulatory moiety, such as a protein that modulates othertypes of immune response, eg an anti-cytokine agent or a cytokinereceptor blocker.

It is preferred that the cleavage site of the therapeutic reagent isenzyme based.

The cleavage site may comprise a polypeptide/amino acid sequence in theprodrug susceptible to a specific enzymatic cleavage. The enzyme is thatpresent at sites of inflammation or immuno pathology, for examplesmatrix metalloproteinases (MMPs) and/or aggrecanases. The cleavagesite(s) in the therapeutic reagent can be cleaved by specific enzymessuch as MMPs and aggrecanases at the target site to release the CReg, orother active agent acting as an immunoregulatory moiety, from thecarrier protein, such as an Ig Fc domain, in order to restore functionof the active agent.

In a preferred aspect, the therapeutic reagent comprises a cleavage sitethat itself comprises a polypeptide/amino acid sequence incorporatedbetween the regulatory moiety/CReg and the hinge region of an Ig, thecleavage site being susceptible to cleavage by one or more enzymes,selected from: MMP3, MMP8 and other members of the MMP family, and thoseof the aggrecanase family. Examples of such are mentioned by Mercuri etal in J Biol Chem 274 32387 (1999). Several publications describe thepreparation of recombinant aggrecanase, such as Tortorella et al inScience 284 1664 (1999) [aggrecanase-1] and Horber et al in Matrix Biol19 533 (2000) Other enzymes expressed specifically or in increasedabundance at the target site may also be utilised, with appropriatemodification of the cleavage site.

In the Examples hereinbelow, a preferred cleavage sequence is a part ofthe inter-globular-domain (IGD) of aggrecan, which comprisesapproximately 120 amino acids. Ideally said cleavage sequence comprisesthe minimum number of amino acids needed for cleavage to occur. It ispreferred that the cleavage sequence for aggrecanase or MMPs comprisesin the range of from 17-75 amino acids. More particularly, the cleavagesequence may comprise the aggrecanase IGD cleavage site itself.

Preferred MMP or aggrecan cleavage amino acid sequences comprise: IGD1having the amino acid sequence RNITEGEARSVILTVK; IGD2 having the aminoacid sequence TTFKEEEGLGSVELSGL; and IGD75 having the amino acidsequence: GYTGEDFVDIPENFFGVGGEEDITVQTVTWPDMELPLPRNITEGEARGSVILTVKPIFEVSPSPLEPEEPFTFAP.

It is preferred that the therapeutic reagent is functionallysubstantially inactive prior to cleavage. By ‘substantially inactive’ ismeant that the therapeutic reagent has no, or at least a much reducedability to act on the host, compared to when the regulatory moiety is inits free and/or solubilised state. In this state, the therapeuticreagent can therefore be described as a prodrug. In the case of thetherapeutic reagent comprising a CReg, the reagent has a significantlyreduced ability to act on the host's C system compared to when the CRegis not bound to carrier. For example, at least an order of magnitudereduction in activity can be observed, such as in the range of from a10- to 60-fold reduction. When comparing molecules on a ‘moles of CReg’basis (ie taking into account the mass of the Ig domains), not by massof reagent, then:

-   (1) Rat DAF-IgG1 has a 10-fold decrease in ability to regulate the    classical pathway of complement, compared to soluble DAF (comprising    four short consensus repeats [SCRs]);-   (2) Rat CD59-IgG1 has a 35-fold decrease in ability to regulate the    terminal pathway of complement, compared to free soluble CD59;-   (3) Human DAF-IgG2 or DAF-IgG4 has a 10-fold decrease in activity    (classical pathway), compared to soluble DAF (four SCRs); and-   (4) Human DAF (3 amino terminal SCR)-IgG2 has a 60-fold decrease in    ability to regulate classical pathway, compared to soluble DAF    (three SCRs).

When cleavage occurs, the therapeutic reagent is activated, by releaseof the immunoregulatory moiety/CReg from the carrier protein, so that,in the case of the CReg, for example, it can participate in the controlof C. The principle applies equally to other immune regulators deliveredas Ig fusion proteins. By ‘activated’, we therefore mean that theactivity of the CReg or other active agent is more or less equivalent tothat of the agent in its non-prodrug-bound/soluble form.

For human DAF, in particular, it has been noted that the choice ofantibody isotype greatly influences flexibility at the hinge region ofthe DAF-Ig fusion protein, which in turn influences the activity of theDAF in vitro and in vivo. Altering the activity of the regulatory moietycan be used to control its effect on the host when circulating in thebody. Fusion to either IgG2 or IgG4 Fc domains has the most restrictiveeffect on function of DAF. This is likely to be due to steric hindrancearound the hinge region. Similar principles will apply to the design ofother prodrugs to provide reagents with markedly restricted function butthat are activated upon removal of the Fc by cleavage.

It is envisaged that the cleavage site is positioned between theregulatory moiety/CReg and a joining or hinge region of the carrierprotein/antibody.

Preferably, the therapeutic reagent includes the minimal portion of theCReg or other agent necessary for function upon release.

A further embodiment of the invention relates to a targetabletherapeutic reagent comprising a regulatory moiety-carrier proteinprodrug as described above, in which one of the Fab arms of the Ig isreplaced by a CReg or other immune regulatory molecule and the other bya targeting moiety comprising either a Fab or another protein thatconfers specific binding in the target tissue.

Accordingly, the present invention further provides a therapeuticreagent to control one or more pathologies in a host, said reagentcomprising a immunoregulatory moiety and a carrier protein,characterised in that there is a cleavage site between theimmunoregulatory moiety and the carrier protein, whereby, when theimmunoregulatory moiety is at or adjacent a target in the host, cleavageof the therapeutic reagent occurs at the cleavage site, freeing theregulatory moiety from the carrier protein, wherein the therapeuticreagent is combined with a tissue or cell-specific targeting moiety.

Preferably, the targeting moiety is one or more membrane targetingmolecule(s). These enable the therapeutic reagent to be localised tocell membranes. For example, a targeting moiety may comprise anaddressin (described below) which is incorporated into the therapeuticreagent between the regulatory moiety and the cleavage site such that,following cleavage, the regulatory moiety and the addressin are releasedin a bound form, and the addressin is thus able to direct, or target,the regulatory moiety to a cell membrane.

For example, in the case of CReg-Ig fusion proteins, incorporation of amembrane-targeting molecule can yield therapeutic reagents that haveminimal systemic anti-C activity, and that can bind to membranes butonly become active when released at sites of expression of the relevantenzymes. Membrane targets might include adhesion molecules, or Cfragments deposited in and around inflamed tissue.

As mentioned, membrane targeting may involve the engineering of amyristate group together with a stretch of negatively charged aminoacids into the protein, termed an ‘addressin’ (Smith & Smith in MolImmunol 38 249-55 (2001)). Together, these modifications confer upon theprotein the propensity to associate with lipid membranes throughinsertion of the myristate and charge interactions of the amino acidswith negatively charged phospholipid headgroups. Just one example of aCReg modified with an addressin is APT070, which comprises the threeamino-terminal SCR of CR1 attached to an addressin at the carboxyterminus. Anti-C prodrugs modified in this way will bind lipid membranesand subsequent enzymatic cleavage will release active C regulator attissue site, or visa versa. Additional targeting strategies may includethe sLe^(x) carbohydrate moiety, a ligand for E- and P-selectins onactivated endothelia.

Yet a further embodiment of the invention relates to DNA coding for atherapeutic reagent as described above. In particular, the inventionprovides a method for preparing such a therapeutic reagent, which methodcomprises ligating DNA molecules each encoding the regulatory moiety,the carrier protein and the cleavage site comprising the therapeuticreagent and giving expression to the DNA sequence therby encoding thetherapeutic reagent.

Preferably, the therapeutic reagent protein is expressed in a eukaryoticsystem using a high expression vector. A preferred expression vector ispDR2ΔEF1α (as described by Charreau in Transplantation 58 1222 (1994)),although other vectors may also be used.

A further embodiment of the invention relates to a culture systemcomprising the cDNA encoding the therapeutic reagent protein asdescribed above inserted in a high expression vector and transfected inCHO cells or other appropriate eukaryotic expression systems, includingDNA encoding a regulatory moiety and a carrier protein, separated by DNAencoding a cleavage site^(˜).

According to a further aspect of the invention there is provided the useof a therapeutic reagent, as aforedescribed, in the preparation of amedicament for the treatment of disease.

A further aspect of the invention includes a method of treating diseasein a host, comprising administering a therapeutically effective amountof a therapeutic reagent according to this invention to the host.

It is preferred that the therapeutic reagent is suitable for treatinghumans and therefore a preferred host is man.

Diseases which may be treated include all those in which complementplays a role in pathology. Such diseases include inflammatory diseases,such as rheumatoid arthritis; immunological disorders eg. Arthusreaction; ischaemic disorders or cancer. Further conditions that may betreated include adult respiratory distress syndrome (ARDS), systemiclupus erythematosis, multiple sclerosis and other demyelinatingdisorders, glomerulonephritis, ischemia-reperfusion injuries, such asstroke and myocardial infarction, myaesthenia gravis, allergic reactionssuch as asthma and dermatological disorders, and rejection intransplantation.

It is envisaged that the therapeutic reagent may be administeredsystemically, such as via the intravenous, intramuscular or subcutaneousroutes. Intravenous administration is particularly applicable wheremultiple sites in the body are involved, as, for example, in autoimmunedisease. In some circumstances, the agent may be injected directly to asite of inflammation, such as intra-articularly in an inflamed joint inarthritis.

According to a further aspect of the invention there is provided apharmaceutical composition including the therapeutic reagent, asaforedescribed, which is combined with a pharmaceutically acceptablecarrier, which carrier comprises those conventionally known in the art.

Although the invention has been described with particular reference tocertain CRegs it is envisaged that the invention may apply to otherregulators of the immune response, where the regulatory moiety is, forexample, a molecule that can have immunoregulatory effects on the body.The reagents could be used for a range of diseases for both human andveterinary applications.

The invention will now be illustrated by the following Examples, inwhich reference is made to the accompanying Figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of new studies of in vivo half-life of DAF-Igand soluble DAF (sDAF). Radiolabelled DAF-Ig (●) or sDAF (∘) wasadministered to rats, blood was removed at certain timepoints andprotein bound radioactivity was determined. Results are expressed aspercent of levels at 3 minutes and represent the means of fiveanimals±SD.

FIG. 2 illustrates the therapeutic effect of DAF-Ig on antigen inducedarthritis, a rat model of rheumatoid arthritis. Methylated BSA wasintroduced into the right knee of immune rats. DAF-Ig (∘) or salinecontrol (●) was administered to the joint at the same time. Swelling ofthe joint was measured daily and compared to that of the left knee.Results represent the mean of five animals±SD. These results show areduction in swelling and disease severity in treated compared withcontrol animals from day 2 onwards. *p<0.01, **p<0.001

FIG. 3 shows new studies concerning in vitro complement regulatoryfunction of DAF-Ig and the effect of cleavage by the enzyme, papain,that cleaves the DAF-Ig and releases active DAF. a) Antibody sensitisederythrocytes were incubated in GVB with rat serum and differentconcentrations of sCRI (□), DAF-Ig (Δ), sDAF (∇) or a non-regulatory Igfusion protein (◯). b) shows results after treatment with papain.Haemolysis was assessed by release of haemoglobin to the supernatant andpercent lysis was determined. Results represent the mean value±SD ofthree determinations.

FIG. 4 a) shows the results of new studies concerning the in vitrocomplement regulatory function of CD59-Ig and the effect of using aspacer in CD59 fusion proteins. Guinea pig erythrocytes bearing C5b-7sites were incubated in PBS/EDTA with rat serum and differentconcentrations of test protein. Results represent the mean value±SD ofthree determinations showing the functional comparison of CD59-Ig (□),CD59-spacer-Ig (⋄), a non-regulatory Ig fusion protein (◯) and sCD59(●). b) shows results after treatment with papain. As seen, cleaved CD59activity is comparable with sCD59 activity.

FIG. 5 shows an example of the results of a haemolytic assay showing theability of different human DAF-Ig fusion proteins (DAF-G2, DAF-G4,S3-G4, S3-G2) to inhibit complement, compared with inhibition achievedby sCR1 and soluble DAF with no Fc attached. DAF-G2, DAF-G4, four SCR ofDAF attached to Fc of IgG2 and IgG4 respectively; S3-G2, S3-G4, threeSCR of DAF attached to Fc of IgG2 and IgG4 respectively; SCRs1-3,SCRs1-4, soluble DAF with three and four SCR domains respectively and noFc.

FIG. 6 shows a schematic representation of a therapeutic reagentaccording to the invention having CReg and IgFc moieties, together witha cleavage site.

FIG. 7 shows the portions of IGD of aggrecan (from 17-75 amino acids)incorporated into DAF-Ig of the invention, between the antibody hingeand DAF. Single underlined: major MMP cleavage site (including MMP3 andMMP8). Double underlined: major aggrecanase cleavage site, also cleavedby MMP8. Dotted underlined:alternative aggrecanase cleavage site.

FIG. 8 DNA sequences encoding different lengths of the IGD of aggrecanwere cloned into the expression vector between human DAF and human IgG4hinge. Lane (1) no IGD, (2) IGD 1 (3) IGD 2 (4) a control ‘scrambled’polypeptide sequence (5) 75 amino acids of IGD. Supernatent fromexpressing cells were subject to SDS-PAGE and Western blot. Blots wereprobed with (a) anti-human Fc or (b) anti-human DAF.

FIG. 9 Human DAF-Ig containing 75 amino acids of IGD was purified byprotein A affinity chromatography and subjected to SDS-PAGE. The lowerband represents DAF-Ig which is not disulphide bonded at the hinge, thisis characteristic of human IgG4. Gel filtration studies indicate thatthe ‘half-forms’ are linked through non-covalent bonds undernon-denaturing conditions.

FIG. 10 The prodrug shown in FIG. 9 (1.5 μg ) was digested with MMP3 orMMP8 and subjected to non-reducing SDS PAGE and Western blot. Blots wereprobed with polyclonal anti-human DAF. Lane 1: no enzyme; Lanes 2-4:414, 138 and 46 ng cdMMP3, respectively; Lanes 5-7; 300, 100 and 37 ngMMP8 respectively.

FIG. 11 shows a schematic of the cleavage sites on a therapeutic reagentfollowing cleavage of the prodrug described in the invention, detectedby anti-neoepitope antibodies. BC3, new N-terminus at site 1; BC4, newC-terminus at site 3; BC14, new N-terminus at site 3.

FIG. 12 shows a prodrug as shown in FIG. 6, digested with MMP8 oraggrecanase and subjected to reducing SDS PAGE and Western blot.Anti-neoepitope antibodies were used to detect the new N-terminifollowing cleavage as described above.

FIG. 13 shows detection of the new C-terminus of a prodrug as shown inFIG. 6, following cleavage at the major MMP site.

FIG. 14 shows cleavage of DAF (4 SCRs)-IGD75-IgG4 with MMP3 and MMP8using silver stained SDS PAGES gels.

FIG. 15 shows cleavage of DAF (4 SCRs)-IGD1-IgG4 with MMP8 using silverstained SDS PAGE gels.

FIG. 16 schematically shows human S3-DAF-Ig2 (three SCR of DAF attachedto IgG2 Fc) incorporating a DIPEN cleavage site.

FIG. 17 graphically shows the results of functional tests using theprodrug of FIG. 16.

FIGS. 18 to 22 show results of tests on the prodrug of FIG. 16 asfollows.

-   FIG. 18: gel filtration to purify.-   FIG. 19: resistance of parent molecule to MMP3 cleavage at 37° C.    and stability of prodrug (no degradation) at 37° C.-   FIG. 20: cleavage of prodrug by incubation with MMP3 at various    doses at 37° C. for up to 24 hours.-   FIG. 21: detection of neoepitopes following cleavage by MMP3.-   FIG. 22: detection with anti-DAF mAb of release of DAF from prodrug.

FIG. 23 graphically shows restoration of function and inhibitoryactivity of the prodrug versus controls. Controls: comprised the prodrugwithout MMP3. Inset: gel to illustrate cleavage of prodrug by MMP3 at 3,6 and 24 hours.

FIG. 24 comprises three sets of results (A B and C) on the prodrugcomprising 4 SCR of DAF, 75 amino acids of IgD and IgG4 Pc, to determinewhether enzyme released from chondrocytes treated with inflammatorycytokines, rather than purified enzyme, could cleave the prodrug. As canbe seen, in each instance, the enzymes are effective and the prodrug isactivated.

EXAMPLES

Preparation of Recombinant Proteins

Method Example 1 Rat DAF-Ig and CD59-Ig

DNA encoding the four SCRs of rat DAF was cloned into the expressionvector SigpIg (R&D Systems) and that encoding the signal peptide andentire extracellular domain of CD59, omitting the GPI anchor signalsequence, was cloned into the. vector pIgPlus (R&D Systems). In bothcases DNA encoding the regulator was cloned upstream of and in framewith DNA encoding the hinge and Fc Domains of human IgG1. In order toachieve high levels of expression, DNA encoding the signal peptide,regulator and Ig domains was then sub-cloned using PCR into the highexpression vector pDR2ΔEF1α. CHO cells were transfected usinglipofectamine (Life Technologies) according to the manufacturer'sinstructions and stable lines were established by selection with 400μg/ml Hygromycin B (Life Technologies). Supernatant was collected andpassed over a Prosep A column (Bioprocessing Ltd, Consett, UK) to purifythe fusion protein. The column was washed with PBS and with 0.1M citratebuffer pH5.0 to remove contaminating bovine Ig and the fusion proteinwas eluted with 0.1M Glycine/HCl pH2.5. Eluted protein was neutralisedwith Tris, concentrated by ultrafiltration and dialysed into PBS.

Method Example 2 Soluble Rat DAF and soluble CD59

DNA encoding the signal peptide and four SCRs of rat DAF (C-terminalresidue Lys254) was cloned directly into the expression vectorpDR2ΔEF1α. CHO cells were transfected as described above. sDAF wasprepared by affinity chromatography on a monoclonal anti-DAF (RDII-24)column. Protein was eluted using 50 mM diethylamine pH11 and immediatelylyophilised. The dried protein was solubilised in phosphate buffer with1M NaCl and was applied to a Superose 12 gel filtration column(Amersham-Pharmacia Biotech AB, Uppsala, Sweden). Proteins were elutedwith PBS and fractions containing DAF were identified. The pure DAF wasconcentrated by ultrafiltration. Soluble CD59 containing the entireextracellular portion (omitting the GPI anchor) was also produced intransfected CHO cells and was obtained from idENTIGEN^(cyf) (Cardiff,UK).

Method Example 3 Control SCR Fusion Protein

A control SCR-containing fusion protein was also prepared in anidentical manner to that of Example 1. This protein had no C-regulatoryfunction.

Method Example 4 CD59-pacer-Ig According to the Invention

A CD59-containing fusion protein was also prepared in which the aminoacids (Ser-Gly-Gly-Gly-Gly)₂-Ser were inserted between CD59 and theantibody hinge using two stage PCR. Briefly, DNA encoding CD59 and theIg domains was reamplified in two separate reactions using new primersthat incorporated the sequence of the spacer domain at the 3′ end ofCD59 and at the 5′ end of the Ig hinge. The two PCR products were mixedtogether and allowed to anneal at complementary DNA sequences encodingthe spacer domain. Following PCR using outside primers, the product wasligated into the expression vector pDR2ΔEF1α. Cells were transfected andthe second CD59-Ig protein was purified as described above. Proteinconcentrations were determined using Pierce Comassie assay (PerbioScience UK Ltd, Tattenhall, UK) using bovine serum albumin as astandard.

CD59-Ig has a mass of 77 Kda, CD59-spacer-Ig has a mass of 78.5 Kda andDAF-Ig has a mass of 122 Kda, these masses being confirmed by massspectrometry.

Method Example 5 Human DAF-IgG2 and Human DAF-IgG4 According to theInvention

Human DAF-IgG1 was generated as described by Harris C L et al. (2000),Immunology, 100, 462. Fusion proteins consisting of human DAF and eitherIgG2 or IgG4 hinge were generated as follows:

DNA encoding the hinge and Fc of human IgG4 or IgG2 were amplified byRT-PCR from human peripheral blood lymphocyte RNA. The amino terminalsequences of the antibody hinges are as follows:

-   IgG4 short hinge: KYGPPC . . .-   IgG4 long hinge: VDKRVES . . .-   IgG2: ERKCCV . . .

Primers incorporated restriction sites to enable later ligation into ahigh expression vector pDR2ΔEF1α ((BamH1 at the 5′ end and EcoRV at the3′ end). DNA encoding the signal peptide and either the first three orfour SCR domains of hDAF was amplified by PCR using plasmid containingDAF sequences as a template. The carboxy-terminal sequences of the DAFdomains are as follows:

-   -   3SCR form: . . . PECREIY    -   4SCR form (with IgG4 long hinge): . . . KSLTSK    -   4SCR form (with IgG4 short hinge and IgG2): . . . PPPECRG

Amplified DNA was separated from the template by electrophoresis on anagarose gel. The insert was extracted from the gel, cut with suitablerestriction enzymes at sites encoded on the PCR primers (BamH1 at the 3′end and Xbal at the 5′ end) and ligated into pDR2ΔEF1α upstream of andin frame with DNA encoding the hinge and Fc domains of the humanimmunoglobulin. DNA proof-reading polymerase was used in the PCRreactions and sequencing confirmed that no errors had been introduced byPCR. CHO cells were transfected using lipofectamine (Life Technologies)according to the manufacturer's instructions and stable lines wereestablished by selection with 400 μg/ml Hygromycin B (LifeTechnologies). Supernatant was collected and passed over a Prosep Acolumn (Bioprocessing Ltd, Consett, UK) to purify the fusion protein.The column was washed with PBS and with 0.1M citrate buffer pH5.0 toremove contaminating bovine Ig and the fusion protein was eluted with0.1M Glycine/HCl pH2.5. Eluted protein was neutralised with Tris,concentrated by ultrafiltration and dialysed into PBS. The purifiedproteins were analysed by SDS PAGE.

Functional Assay Protocols

Protocol Example 1 Functional Analysis of DAF-Ig and sDAF

In order to assess function of rat DAF, antibody coated sheeperythrocytes (E; 2% (v:v)) were prepared by incubating cells in PBS for30 minutes with 1/500 dilution of rabbit anti-sheep E (Amboceptor,Behring Diagnostics GmbH). Sensitised E were washed three times in GVB(Gelatin Veronal Buffer comprising CFD [C-fixation Diluent with added0.1% (w/v) gelatin (Immunol 100 463 (2000)]) and re-suspended to 2%. Inorder to determine a concentration of rat serum giving partial lysis(50-80%), antibody coated sheep E (EA) were incubated for 30 minutes at37° C. with different dilutions of serum. Following pelleting of cellsby centrifugation, amount of lysis was quantitated by adding an aliquotof supernatant (50 μl) to water (100 μl) and measuring absorbance at 415nm. Control samples were prepared by adding buffer only (0% control) or0.1% Triton X100 (100% control) to the E instead of serum. % lysis wascalculated as follows: % lysis=100×(A415 sample−A415 0% control)/(A415100% control−A415 0% control). To test function of the recombinantinhibitors, EA were incubated with the predetermined dilution of ratserum giving 50-80% lysis and different dilutions of the test protein.Following incubation at 37° C., % lysis was determined as describedabove.

Protocol Example 2 Functional Analysis of CD59

Guinea pig E(GPE) were washed and resuspended in GVB at 2% (v:v). Thesewere incubated for 30 minutes at 37° C. with an equal volume of 25%(v:v) normal human serum from which C8 had been depleted be passage overa monoclonal anti-C8 affinity column. The resulting cells (GPE-C5b7)were washed and re-suspended at 2% in PBS.10 mM EDTA The amount of ratserum giving 50-80% lysis was determined by incubating GPE-C5b7 for 30minutes at 37° C. with dilutions of rat serum in PBS/EDTA. In order toassess function of soluble CD59, GPE-C5b7 were incubated in PBS, EDTAwith dilutions of the test reagent and the predetermined concentrationof rat serum. 0% and 100% controls were included and % lysis wasdetermined as described above.

Illustrative Results of Known Studies

Illustrative Example 1 In Vivo Clearance of DAF-Ig is Slowed Compared tosDAF

In order to study the effect of the Fc domain (immunoglobulincrystallisable fragment) on clearance of soluble DAF (sDAF), DAF-Ig andsDAF were radiolabelled with ¹²⁵I. Animals were administered with asingle dose of either reagent and samples of blood were removed atcertain timepoints. Protein was precipitated using TCA and protein boundcounts were measured in a gamma counter. At 1 hour followingadministration, sDAF levels were down to 20% of that seen at 3 minutes,DAF-Ig levels were still 80% of those at 3 minutes, demonstrating theenhancement of half life as a consequence of fusion to Ig domains (FIG.1).

Illustrative Example 2 DAF-Ig Delays Onset and Inhibits Progression inAntigen Induced Arthritis (AIA)

AIA was induced in Wistar rats (Goodfellow et al, Clinical andExperimental Immunology (1997), 110, 45). Briefly, methylated BSA (mBSA)was introduced into the right knee of five rats pre-immunised with mBSA;0.45 mg DAF-Ig or the same volume of saline (control animals) wasincluded with the antigen. Disease progression was monitored bycomparing swelling of the right knee to that of the left, over thecourse of a week. Rat DAF-Ig caused a significant reduction in swellingand disease severity compared to control animals from day 2 onwards(FIG. 2). These results indicate the long-term effects of DAF-Ig fusionproteins in vitro.

Results of New Studies Relating to the Invention

Example 1 In vitro Functional Analyses of DAF-Ig, sDAF, CD59-Ig andCD59-Cleavage by Papain

The ability of DAF-Ig and sDAF to inhibit the classical pathway of C wasanalysed using a haemolysis assay and was compared to inhibition oflysis achieved with sCR1. Both sDAF and sCR1 were powerful inhibitors oflysis, while DAF-Ig showed a reduced ability to inhibit lysis (FIG. 3).Tests using papain to cleave Ig from DAF indicated that the functionalactivity of DAF in vitro could be restored by removal from the Ig. Thiswas shown by the ft that DAF released from Ig domains by digestion withpapain, had identical activity to sDAF secreted from CHO cells.

The ability of CD59-Ig and CD59-spacer-Ig to inhibit C was also testedusing haemolysis assays specific for the terminal pathway. Again, thefusion protein showed a much lowered ability to inhibit MAC formationwhen compared to CD59 released from CD59-Ig using papain. This, like theDAF analysis, indicated that cleavage of the Ig from the CD59 increasedactivity (FIG. 4). Further, the presence of the spacer domain indicatedits ability to modify the activity of the regulatory moiety, andimplicated steric factors in loss of regulatory function in the prodrug.The presence of a spacer domain enhanced regulatory function of CD59-Igalthough activity was still low compared to sCD59.

FIGS. 3 and 4 show that both DAF-Ig and CD59-Ig had less complementregulatory capacity than the soluble forms lacking the Fc. It is likelythat this is due to steric constraints in which the active site of theregulatory proteins cannot access and bind the large multimolecularsubstrate, be it the C3/C5 convertase or MAC. This is supported by theobservation that enzymatic removal of the Fc domains restores fillfunction to the released regulatory protein.

In addition to modification of activity of a therapeutic reagent using aspacer domain as described above and shown on FIG. 4, the type ofantibody used as the carrier protein can influence the effect of atherapeutic reagent. This is presumably due to the steric influence ofvariations in the hinge region of an antibody. Studies as shown in FIG.5, indicate that Ig with less flexible hinge regions, for example IgG2,cause more restriction on the functional activity of a linkedtherapeutic reagent in vitro. Deletion of the fourth SCR of human DAFfurther restricted functional activity of the CReg (FIG. 5).

Example 2 Preparation of New Fusion Proteins Incorporating IGD1, IGD2and IGD75 Cleavage Sites

FIG. 6 shows a diagrammatic representation of a prodrug form of atherapeutic reagent according to the invention, showing the position oftargeted enzyme cleavage sites between a regulatory moiety and the hingeregion of a carrier protein.

In this Example, studies were made using cleavage sites appropriate toenzymes involved in inflammatory disease such as arthritis. MMPs andaggrecanases are involved in inflammation of the joints and they destroycartilage by proteolysis of the major constituent proteoglycan,aggrecan. Polypeptides containing three different cleavage sites forsome of these enzymes (MMP3, MMP8 and aggrecanase (ADAM-TS4)) wereincorporated into CReg-Ig fusion proteins between the CReg and thehinge.

The length of the polypeptide was restricted to 17 amino acids each(termed here IGD1 and IGD2, each incorporating a different cleavagesite) or 75 amino acids (termed here IGD75, incorporating two cleavagesites: the site from IGD1 and another site). Scrambled IGD is a controlsequence containing no cleavage sites. (Sequences shown in FIG. 7).

In the case of IGD1, the amino acid sequence is RNITEGEARGSVILTVK; IGD2has the amino-acid sequence TTFKEEEGLGSVELSGL; and IGD75 has the aminoacid sequence:GYTGEDFVDIPENFFGVGGEE-DITVQTVTWPDMELPLPRNITEGEARGSVILTVKPIFEVSPSPLEPEEPFTFAP.

To incorporate the enzyme sites, complementary DNA oligomers encodingthe short IGD sites with suitable restriction sites at both ends wereused (BAMH1). These were annealed together, restricted with BamH1 andligated into the expression vector between DNA encoding DAF and theantibody hinge. The longer stretch of DNA encoding 75 amino acids of IGDwas amplified using PCR from a plasmid template and similarly ligatedinto the vector at the BamH1 site (primers incorporated the restrictionsite). CHO cells were transfected as described above and the culturesupernatant was collected. FIG. 8 shows Western Blot analysis usinganti-human Ig (goat anti-human Fc-HRPO conjugated; 1:1000 dilution,available from Sigma) to demonstrate the presence of DAF-Ig in culturesupernatant which contained target enzyme cleavage sites. In the case ofaggrecan, several portions of the IGD can be included. FIG. 9 shows aSDS-PAGE analysis of the purified DAF-Ig prodrug. FIG. 10 showsdigestion of the DAF-Ig prodrug with MMP3 and MMP8 enzymes and detectionof released DAF in a western blot.

A schematic showing various cleavage sites of a therapeutic reagentaccording to the invention and their detectability by anti-neo-epitopeantibodies is shown in (FIG. 11).

Secreted fusion proteins were purified by Protein A affinitychromatography. The fusion protein containing IGD 75 was furtherpurified by gel filtration on a Superose 12 gel filtration column(Amersham-Pharacia Biotech AB), equilibriated with 50 Mm Tris pH 7.5,100 mM NaCl, 10 mM CaCl₂.2H₂0 (FIG. 9). The eluted protein was digestedwith MMP3, MMP8 and aggrecanase (FIG. 10).

The therapeutic reagent (3.5 μg) was incubated at 37° C. for 24 hourswith 0.3 μg neutrophil MMP8 (Calbiochem) or with recombinantaggrecanase. The sample was lyophilised, re-dissolved in reducing SDSPAGE loading buffer and subjected to SDS PAGE and Western blot (1 μgprotein/lane). The blots were probed with anti-neoepitope antibodiesthat recognise the ‘cut-ends’ of aggrecan. Primary antibodies weredetected with HRPO-linked secondary antibodies and bands were visualisedusing enhanced chemiluminescence (ECL). The antibody BC3 (Hughes et alBiochem J 305 700 (1995)) recognises the new N-terminus formed followingcleavage at the major aggrecanase cleavage site (site 1 in FIG. 11); theantibody BC14 (Caterson et al in Acta Orthop Scand Suppl 266 121 (1995))recognises the new N-terminus formed following cleavage at the major MMPsite (FFG—site 3 in FIG. 11). BC13 recognises the new C-terminusfollowing cleavage at the major aggrecanase site (EGE). BC4 recognisesthe new C-terminus created following cleavage at the major MMP site(PEN—site 3 in FIG. 7). The antibodies were used to probe the blots at1:100 (tissue culture supernatant). Cleavage of the prodrug by MMP8 atboth enzymes sites was detected (FIG. 12, lanes 1 and 2) and alsocleavage by aggrecanase at the aggrecanase site (FIG. 12, lane 3).

The therapeutic reagent in prodrug form was digested with MMP8 andanalysed by Western blot as described above. The blot was probed with ananti-neo-epitope antibody that recognises the new C-terminus formedfollowing cleavage at the major MMP site, BC4 recognises the newC-terminus formed following cleavage at the major MMP site (PEN—site 3);this protein fragment comprises hDAF and a small stretch of aggrecan IGD(FIG. 13).

The results shown in FIGS. 11 to 13 demonstrate that short enzyme sitescan be incorporated into Ig-fusion proteins and that the active agentcan be released following cleavage by the target enzyme. FIG. 14 shows asilver-stained SDS-PAGE analysis of the cleavage by MMP8 and MMP3 of theprodrug comprising DAF attached to the IgG4 Fc via the IGD75 linker(FIG. 7). FIG. 15 shows a silver-stained SDS-PAGE analysis of thecleavage by MMP8 of the prodrug comprising DAF attached to the IgG4 Fcvia the IgD1 linker (FIG. 7).

Example 3 Generation of S3-DAF-IgG2 Prodrug Containing ‘DIPEN’ EnzymeSite

DNA encoding the hinge (starting amino acids ERKCCV . . . ), CH2 and CH3domains of human IgG2 was amplified by RT-PCR from peripheral bloodmononuclear cell total RNA and ligated into the high expression vectorPDR2ΔEF1α (Charreau et al in Transplantation 58 1222 (1994). DNAencoding the signal peptide and first three SCR of human DAF (finishingamino acids . . . CREIY) was amplified by PCR from plasmid template andligated into the vector upstream of and in frame with DNA encoding theantibody hinge, as described in Method Example 5. CHO cells weretransfected and fusion protein (S3 DAF-IgG2; also termed ‘parent’molecule, not having cleavage site) was purified as described in MethodExample 1. A prodrug form (termed the ‘DIPEN prodrug’) of the reagentwas prepared as described in Example 2 using BamH1 restriction sites byinserting DNA (purchased oligomers) encoding the enzyme site here termed‘DIPEN’ between DNA encoding DAF and the Ig. The sequence of theinserted enzyme site is GEDFVDIPENFFGVGGEED; this is illustrated in FIG.16 where the cleavage site is indicated (immediately upstream of theDIPEN sequence). This site is cleaved by most MMPs.

Functional activity of S3 DAF-IgG2 and Corresponding DIPEN Prodrug

S3 DAF-IgG2 (parent) and the DIPEN prodrug were purified by protein Aaffinity chromatography and gel filtration on a Superose 12 column asdescribed in Example 2. Functional activity was assessed by inhibitionof lysis of antibody coated sheep erythrocytes (EA) essentially asdescribed in Protocol Example 1, ensuring that the buffer composition(GVB, PBS or Tris/Ca²⁺) was equivalent in each incubation (FIG. 17).Control incubations included a non-regulatory SCR-containing fusionprotein (negative control) and a three SCR form of DAF produced inyeast. Calculation of IH₅₀ and adjustment for molarity (equivalent molesof DAF) indicated that the DIPEN prodrug was approximately 20 fold lessactive than the three SCR form of DAF. Incorporation of the enzyme siteacted as a ‘spacer’ domain and restored some activity to S3 DAF-IgG2(compare ‘parent’ to DIPEN prodrug).

Stability of S3 DAF-IgG2 and DIPEN Prodrug

Both reagents were gel filtered into Tris/NaCl/Ca²⁺ as described inExample 2 (FIG. 18, filtration of prodrug), and incubated for 24 hoursat 37° C. S3 DAF-IgG2 was incubated in the presence of MMP3 (Calbiochem,recombinant catalytic domain) to assess non-specific cleavage by thetarget enzyme. Aliquots were removed from the incubations at thespecified time-points and stored frozen in reducing SDS PAGE loadingbuffer until the end of the experiment. Samples were run on a 10% geland silver stained according to the method of Morrissey in Anal Biochem117 307-10 (1980) (FIG. 19). Both molecules were stable stored at 37°C., the parent molecule was also stable in the presence of MMP3.

Cleavage of DIPEN Prodrug with MMP3

The prodrug was incubated for 1, 2.5, 5, 7.5 and 24 hours with MMP3 atthe concentrations specified in the following table. Prodrug (μg/ml)MMP3 (μg/ml) Ratio Prodrug:MMP3 100 10 10:1 100 2 50:1 200 2 100:1 

Aliquots of each incubation were analysed by silver staining asdescribed above. The prodrug was cleaved by MMP3 even at ratios of 100:1(w:w) (FIG. 20). The upper cleavage band (≈35 kDa) represents thereleased Fc domains (confirmed by Western blot); the lower cleavageproduct (≈30 kDa) is the released DAF (three SCRs). The DIPEN prodrugwas also cleaved by MMP8 (not shown) and could therefore form a targetfor a multitude of metalloproteases.

Western Blot Detection of Released DAF and Neo-Epitope Formation

In order to confirm that the prodrug had been cleaved at themetalloprotease site, portions of the incubations described above (FIG.20) were run on an 11% reducing SDS PAGE gel, Western blotted and probedwith BC14 to detect the neo-epitope (antibody ‘side’) formed followingcleavage (as described in Example 2 and FIG. 11). BC14-reactiveneo-epitope was detected following incubation of DIPEN prodrug withMMP3, but not when the prodrug was incubated alone (prodrug control) orwhen the parent molecule was incubated with MMP3 (FIG. 21). Samples froma similar incubation (DIPEN prodrug at 200 μg/ml, MMP3 at 5 μg/ml) wereanalysed by non-reducing SDS PAGE and Western blot using a monoclonalanti-DAF antibody. The prodrug was incubated in the absence of MMP3 as acontrol. Released DAF was detected when the prodrug was incubated withenzyme (FIG. 22).

Restoration of Function Following Cleavage

In order to demonstrate that incubation of the DIPEN prodrug with MMP3restored complement-regulatory activty, the following incubations wereset up:

-   (1) Prodrug (200 μg/ml)-   (2) Prodrug (200 μg/ml); MMP3 (5 μg/ml)-   (3) BSA (200 μg/ml); MM3 (5 μg/ml)

Proteins were incubated for up to 24 hours and analysed by SDS PAGE andsilver stain (inset to FIG. 23). 3 hour and 6 hour incubations ofprodrug (with and without MMP3) and 6 hour incubation of BSA (with MMP3)were analysed by haemolysis assay as described in Protocol Example forcomplement-regulatory activity. Per cent lysis and inhibition (comparedto negative control: non-regulatory fusion protein) were calculated(FIG. 23). MMP3 in the BSA incubation had no effect oncomplement-mediated lysis of EA. As can be seen in FIG. 23, incubationof the DIPEN prodrug with MMP3 for 3 hours restored almost all DAFactivity.

Cleavage of Anti-Complement Prodrug Using Native Enzyme Release fromActivated Chondrocytes

In order to determine whether enzyme released from chondrocytes treatedwith pro-inflammatory cytokines, rather than purified enzyme, couldcleave the prodrug, experiments were carried out using the therapeuticreagent described in Example 2 (comprising 4 SCRs of human DAF fused tohuman IgG4 and ‘IGD75’ as the inserted enzyme site (FIG. 7)).

By a method analogous to that described by Hughes et al in J Biol Chem272 20269 (1997)), pig chondrocytes were embedded in agarose andcultured in the presence of various cytokines (retinoic acid, IL-1,TNF-α) and prodrug. After 4 days, media samples were dialysed againstwater, lyophilised to dryness and reconstituted with reducing SDS-PAGEloading buffer containing 10% (v/v) mercaptoethanol. Samples wereseparated on 10% SDS-PAGE gels, transferred to nitrocellulose membranesand Western blot analysis was performed with anti-neo-epitopeantibodies. BC3 detects cleavage at the aggrecanase site, whereas BC4(Hughes et al Biochem J 305 700 (1995)) and BC14 detect cleavage at theMMP site (FIG. 11).

FIG. 24A illustrate cleavage of the prodrug by aggrecanase released fromcultures stimulated with either retinoic acid, IL-1α or TNF using 3.4 μgof prodrug and anti-ARG (BC3) monoclonal (1:100). Cleavage of theprodrug at the aggrecanase site was only evident in the presence ofstimulatory cytokines.

In addition, FIG. 24B illustrates an IL-1 dose-dependent cleavage byaggrecanase of prodrug, detected using BC3. FIG. 24C illustratescleavage at the MMP site using BC14; cleavage was evident in thepresence of stimulating cytokines.

CONCLUSION

These data illustrates the preparation of a new prodrug, ideally basedon an IgG2 backbone, and containing a short cleavage site formetalloproteases. Whilst insertion of the enzyme site into the ‘parent’molecule restored some function, the prodrug showed a marked reductionin activity compared to released DAF. This enzyme site could be furthertruncated to retain as much inhibition of function as possible in theprodrug; the parent molecule (having no enzyme site) showed almost twologs reduction in function. The DIPEN prodrug was susceptible tocleavage by several metalloproteases tested; release of DAF andformation of neo-epitopes following digestion was demonstrated by silverstain and Western blot. The cleavage reaction was almost complete asassessed by silver stain. Incubation of the prodrug with MMP3 restoredalmost complete complement-regulatory function to the reagent.Importantly, analysis of the cleavage of these reagents using nativeenzyme released from target cells, chondrocytes is given.

Using a culture system, cleavage of DAF-IGD75-IgG4 at both themetalloprotease and aggrecanase site has been demons Cleavage wastriggered using various pro-inflammatory cytokines.

1-33. (canceled)
 34. A therapeutic reagent to control one or morereactions of the immune system in a host, said therapeutic reagentcomprising: i. at least one regulatory moiety that is animmunoregulatory protein (IRP) or a functional fragment thereof; ii. acarrier protein which is an antibody, or a fragment thereof, and whichrenders said IRP inactive or substantially inactive; and iii. positionedtherebetween at least one cleavage site; whereby when the regulatorymoiety is at, or adjacent, a target organ or tissue in the host, saidcleavage site is cleaved, freeing the regulatory moiety from the carrierprotein and restoring its regulatory activity.
 35. A therapeutic reagentthat is inactive systemically comprising: i. at least one regulatorymoiety that has a therapeutic activity as an immunoregulatory agent; ii.a carrier protein which renders said regulatory moiety inactive orsubstantially inactive; and iii. positioned therebetween at least onecleavage site characterised in that said cleavage site is a substratefor a matrix metalloproteinase (MMP) or an aggrecanase; whereby at siteswhere MMP's or aggrecanase are active in the host said cleavage site iscleaved and said regulatory moiety is freed from the carrier protein andso able to perform its therapeutic function.
 36. A therapeutic reagentto control one or more reactions of the immune system in a host, saidtherapeutic reagent comprising: i. at least one regulatory moiety thatis an immunoregulatory protein (IRP) or a functional fragment thereof:ii. a carrier protein which renders said IRP inactive or substantiallyinactive; and iii. positioned therebetween at least one cleavage sitecharacterised in that said cleavage site comprises a substrate for atleast one enzyme of the Complement system; whereby when said therapeuticreagent is at or adjacent a site in the host where Complement is activesaid cleavage site is cleaved so freeing the immunoregulatory moietyfrom the carrier protein and enabling it to perform its immunoregulatoryactivity.
 37. A therapeutic reagent according to claim 34, wherein theimmunoregulatory protein (IRP) is a complement regulatory protein (CReg)or a functional fragment thereof.
 38. A therapeutic reagent according toclaim 36, wherein the immunoregulatory protein (IRP) is a complementregulatory protein (CReg) or a functional fragment thereof.
 39. Atherapeutic reagent according to claim 37, wherein the (CReg) actseither as: a decay accelerating factor, or a cofactor for the plasmaprotease factor I, or to inhibit formation of membrane attack complex,or a combination thereof.
 40. A therapeutic reagent according to claim38, wherein the (CReg) acts either as: a decay accelerating factor, or acofactor for the plasma protease factor I, or to inhibit formation ofmembrane attack complex, or a combination thereof.
 41. A therapeuticreagent according to claim 34, wherein said reagent has more than oneregulatory moiety, two of which have different activities.
 42. Atherapeutic reagent according to claim 35, wherein said reagent has morethan one regulatory moiety, two of which have different activities. 43.A therapeutic reagent according to claim 36, wherein said reagent hasmore than one regulatory moiety, two of which have different activities.44. A therapeutic reagent according to claim 34, wherein said cleavagesite is an enzymatically cleavable site.
 45. A therapeutic reagentaccording to claim 35, wherein said cleavage site is an enzymaticallycleavable site.
 46. A therapeutic reagent according to claim 36, whereinsaid cleavage site is an enzymatically cleavable site.
 47. A therapeuticreagent according to claim 34, wherein said cleavage site is a substratefor an enzyme of the Complement system.
 48. A therapeutic reagentaccording to claim 35, wherein said cleavage site is a substrate for anenzyme of the Complement system.
 49. A therapeutic reagent according toclaim 36, wherein said cleavage site is a substrate for an enzyme of theComplement system.
 50. A therapeutic reagent according to claim 34,wherein said cleavage site comprises a plurality of cleavage sites. 51.A therapeutic reagent according to claim 35, wherein said cleavage sitecomprises a plurality of cleavage sites.
 52. A therapeutic reagentaccording to claim 36, wherein said cleavage site comprises a pluralityof cleavage sites.
 53. A therapeutic reagent according to claim 50,wherein at least one of said cleavage sites comprises a substrate for amatrix metalloproteinase (MMP) or an aggrecanase.
 54. A therapeuticreagent according to claim 51, wherein at least one of said cleavagesites comprises a substrate for a matrix metalloproteinase (MMP) or anaggrecanase.
 55. A therapeutic reagent according to claim 52, wherein atleast one of said cleavage sites comprises a substrate for a matrixmetalloproteinase (MMP) or an aggrecanase.
 56. A therapeutic reagentaccording to claim 53, wherein said cleavage site comprises a substratefor MMP3 or MMP8.
 57. A therapeutic reagent according to claim 54,wherein said cleavage site comprises a substrate for MMP3 or MMP8.
 58. Atherapeutic reagent according to claim 55, wherein said cleavage sitecomprises a substrate for MMP3 or MMP8.
 59. A therapeutic reagentaccording to claim 50, wherein at least one of said cleavage sitescomprises a part of the inter-globular-domain (IGD) of aggrecan.
 60. Atherapeutic reagent according to claim 51, wherein at least one of saidcleavage sites comprises a part of the inter-globular-domain (IGD) ofaggrecan.
 61. A therapeutic reagent according to claim 52, wherein atleast one of said cleavage sites comprises a part of theinter-globular-domain (IGD) of aggrecan.
 62. A therapeutic reagentaccording to claim 59, wherein said cleavage site comprises 17-75 aminoacids of said IGD.
 63. A therapeutic reagent according to claim 60,wherein said cleavage site comprises 17-75 amino acids of said IGD. 64.A therapeutic reagent according to claim 61, wherein said cleavage sitecomprises 17-75 amino acids of said IGD.
 65. A therapeutic reagentaccording to claim 59, wherein the cleavage site comprises the minimalaggrecanase cleavage site in said aggrecan IGD (inter globular domain).66. A therapeutic reagent according to claim 60, wherein the cleavagesite comprises the minimal aggrecanase cleavage site in said aggrecanIGD (inter globular domain).
 67. A therapeutic reagent according toclaim 61, wherein the cleavage site comprises the minimal aggrecanasecleavage site in said aggrecan IGD (inter globular domain).
 68. Atherapeutic reagent according to claim 53, wherein the cleavage siteincludes the amino acid sequence DIPEN.
 69. A therapeutic reagentaccording to claim 54, wherein the cleavage site includes the amino acidsequence DIPEN.
 70. A therapeutic reagent according to claim 55, whereinthe cleavage site includes the amino acid sequence DIPEN.
 71. Atherapeutic reagent according to claim 68, wherein the cleavage siteincludes the amino acid sequence GEDFVDIPENFFGVGGEED.
 72. A therapeuticreagent according to claim 69, wherein the cleavage site includes theamino acid sequence GEDFVDIPENFFGVGGEED.
 73. A therapeutic reagentaccording to claim 70, wherein the cleavage site includes the amino acidsequence GEDFVDIPENFFGVGGEED.
 74. A therapeutic reagent according toclaim 53, wherein the cleavage site includes the amino acid sequenceRNITEGEARGSVILTVK.
 75. A therapeutic reagent according to claim 54,wherein the cleavage site includes the amino acid sequenceRNITEGEARGSVILTVK.
 76. A therapeutic reagent according to claim 55,wherein the cleavage site includes the amino acid sequenceRNITEGEARGSVILTVK.
 77. A therapeutic reagent according to claim 53,wherein the cleavage site includes the amino acid sequenceTTFKEEGLGSVELSGL.
 78. A therapeutic reagent according to claim 54,wherein the cleavage site includes the amino acid sequenceTTFKEEGLGSVELSGL.
 79. A therapeutic reagent according to claim 55,wherein the cleavage site includes the amino acid sequenceTTFKEEGLGSVELSGL.
 80. A therapeutic reagent according to claim 53,wherein the cleavage site includes the amino acid sequenceGYTGEDFVDIPENFFGVGGEEDITVQTVTWPDMELPLPRNITEGEARGSVILTVKPIFEVSPSPLEPEEPFTFAP.


81. A therapeutic reagent according to claim 54, wherein the cleavagesite includes the amino acid sequenceGYTGEDFVDIPENFFGVGGEEDITVQTVTWPDMELPLPRNITEGEARGSVILTVKIPIFEVSPSPLEPEEPFTFAP.


82. A therapeutic reagent according to claim 55, wherein the cleavagesite includes the amino acid sequenceGYTGEDFVDIPENFFGVGGEEDITVQTVTWPDMELPLPRNITEGEARGSVILTVKPIFEVSPSPLEPEEPFTFAP.


83. A therapeutic reagent according to claim 34, wherein the carrierprotein is an antibody, or a fragment thereof.
 84. A therapeutic reagentaccording to claim 35, wherein the carrier protein is an antibody, or afragment thereof.
 85. A therapeutic reagent according to claim 36,wherein the carrier protein is an antibody, or a fragment thereof.
 86. Atherapeutic reagent according to claim 83, wherein said antibody ishuman immunoglobulin IgG4, IgG2 or IgG1.
 87. A therapeutic reagentaccording to claim 84, wherein said antibody is human immunoglobulinIgG4, IgG2 or IgG1.
 88. A therapeutic reagent according to claim 85,wherein said antibody is human immunoglobulin IgG4, IgG2 or IgG1.
 89. Atherapeutic reagent according to claim 83, wherein one or more Fab armsof said antibody or fragment thereof contains, or is replaced by, afurther regulatory moiety.
 90. A therapeutic reagent according to claim84, wherein one or more Fab arms of said antibody or fragment thereofcontains, or is replaced by, a further regulatory moiety.
 91. Atherapeutic reagent according to claim 85, wherein one or more Fab armsof said antibody or fragment thereof contains, or is replaced by, afurther regulatory moiety.
 92. A therapeutic reagent according to claim89, wherein said further regulatory moiety is a targeting moiety.
 93. Atherapeutic reagent according to claim 90, wherein said furtherregulatory moiety is a targeting moiety.
 94. A therapeutic reagentaccording to claim 91, wherein said further regulatory moiety is atargeting moiety.
 95. A therapeutic reagent according to claim 92,wherein the targeting moiety comprises one or more membrane targetingmolecules.
 96. A therapeutic reagent according to claim 93, wherein thetargeting moiety comprises one or more membrane targeting molecules. 97.A therapeutic reagent according to claim 94, wherein the targetingmoiety comprises one or more membrane targeting molecules.
 98. Atherapeutic reagent according to claim 95, wherein the targeting moietycomprises at least one addressin that is incorporated between theregulatory moiety and the cleavage site so that, following cleavage,said addressin directs the regulatory moiety to its target site.
 99. Atherapeutic reagent according to claim 96, wherein the targeting moietycomprises at least one addressin that is incorporated between theregulatory moiety and the cleavage site so that, following cleavage,said addressin directs the regulatory moiety to its target site.
 100. Atherapeutic reagent according to claim 97, wherein the targeting moietycomprises at least one addressin that is incorporated between theregulatory moiety and the cleavage site so that, following cleavage,said addressin directs the regulatory moiety to its target site.
 101. Atherapeutic reagent according to claim 98, wherein the addressin isAPT542.
 102. A therapeutic reagent according to claim 99, wherein theaddressin is APT542.
 103. A therapeutic reagent according to claim 100,wherein the addressin is APT542.
 104. A therapeutic reagent according toclaim 34, wherein the cleavage site is positioned between the regulatorymoiety and a hinge region of the carrier protein.
 105. A therapeuticreagent according to claim 35, wherein the cleavage site is positionedbetween the regulatory moiety and a hinge region of the carrier protein.106. A therapeutic reagent according to claim 36, wherein the cleavagesite is positioned between the regulatory moiety and a hinge region ofthe carrier protein.
 107. A method of making a therapeutic reagentaccording to claim 34, comprising expressing protein from cellstransformed or transfected with at least one nucleic acid moleculeencoding said therapeutic reagent.
 108. A method of making a therapeuticreagent according to claim 35, comprising expressing protein from cellstransformed or transfected with at least one nucleic acid moleculeencoding said therapeutic reagent.
 109. A method of making a therapeuticreagent according to claim 36, comprising expressing protein from cellstransformed or transfected with at least one nucleic acid moleculeencoding said therapeutic reagent.
 110. Use of a therapeutic reagentaccording to claim 34, in the preparation of a medicament for thetreatment of disease.
 111. Use of a therapeutic reagent according toclaim 35, in the preparation of a medicament for the treatment ofdisease.
 112. Use of a therapeutic reagent according to claim 36, in thepreparation of a medicament for the treatment of disease.
 113. Useaccording to claim 110, wherein the disease to be treated includes oneor more of: inflammatory, immunological, traumatic, ischaemic ordisorders in which Complement contributes to pathology such asrheumatoid arthritis, systemic lupus erythematosis, glomerulonephritis,multiple sclerosis, adult respiratory distress syndrome (ARDS),ischemia-reperfusion injury, demyelination, myaesthenia gravis, Arthusreaction or rejection in transplantation.
 114. Use according to claim111, wherein the disease to be treated includes one or more of:inflammatory, immunological, traumatic, ischaemic or disorders in whichComplement contributes to pathology such as rheumatoid arthritis,systemic lupus erythematosis, glomerulonephritis, multiple sclerosis,adult respiratory distress syndrome (ARDS), ischemia-reperfusion injury,demyelination, myaesthenia gravis, Arthus reaction or rejection intransplantation.
 115. Use according to claim 112, wherein the disease tobe treated includes one or more of: inflammatory, immunological,traumatic, ischaemic or disorders in which Complement contributes topathology such as rheumatoid arthritis, systemic lupus erythematosis,glomerulonephritis, multiple sclerosis, adult respiratory distresssyndrome (ARDS), ischemia-reperfusion injury, demyelination, myaestheniagravis, Arthus reaction or rejection in transplantation.
 116. Apharmaceutical composition including a therapeutic reagent according toclaim 34 in combination with a pharmaceutically acceptable carrier. 117.A pharmaceutical composition including a therapeutic reagent accordingto claim 35 in combination with a pharmaceutically acceptable carrier.118. A pharmaceutical composition including a therapeutic reagentaccording to claim 36 in combination with a pharmaceutically acceptablecarrier.
 119. A method of treating an individual comprisingadministering to said individual a therapeutic reagent according toclaim
 34. 120. A method of treating an individual comprisingadministering to said individual a therapeutic reagent according toclaim
 35. 121. A method of treating an individual comprisingadministering to said individual a therapeutic reagent according toclaim
 36. 122. A method of treating an individual comprisingadministering to said individual a pharmaceutical composition accordingto claim
 116. 123. A method of treating an individual comprisingadministering to said individual a pharmaceutical composition accordingto claim
 117. 124. A method of treating an individual comprisingadministering to said individual a pharmaceutical composition accordingto claim 118.